Identification and Expression Patterns of WOX Transcription Factors under Abiotic Stresses in Pinus massoniana

WUSCHEL-related homeobox (WOX) transcription factors (TFs) play a crucial role in regulating plant development and responding to various abiotic stresses. However, the members and functions of WOX proteins in Pinus massoniana remain unclear. In this study, a total of 11 WOX genes were identified, and bioinformatics methods were used for preliminary identification and analysis. The phylogenetic tree revealed that most PmWOXs were distributed in ancient and WUS clades, with only one member found in the intermediate clade. We selected four highly conserved WOX genes within plants for further expression analysis. These genes exhibited expressions across almost all tissues, while PmWOX2, PmWOX3, and PmWOX4 showed high expression levels in the callus, suggesting their potential involvement in specific functions during callus development. Expression patterns under different abiotic stresses indicated that PmWOXs could participate in resisting multiple stresses in P. massoniana. The identification and preliminary analysis of PmWOXs lay the foundation for further research on analyzing the resistance molecular mechanism of P. massoniana to abiotic stresses.


Introduction
Pinaceae plants represent the largest group of tree species among extant gymnosperms, demonstrating strong adaptability in global ecosystems.They play a significant role in carbon-oxygen balance and possess immense economic value [1].Pinus massoniana Lamb.stands out for its remarkable adaptability, and is commonly known as a widespread cultivation tree in southern provinces of China.It serves as a major source of solid wood and pulp production in China.Moreover, the majority of resin and turpentine oil is also produced by P. massoniana [2].However, its growth and resin yield are limited by deteriorating climate, poor growing conditions, and forest pests and diseases.It is very important to analyze the resistance mechanism to various adversities in P. massoniana.
The WOX (WUSCHEL-related homeobox) transcription factor family exists exclusively in plants, and constitutes a subfamily of the homeobox (HB) protein superfamily [3].The WOX family is characterized by a highly conserved domain 'helix-loop-helix-turn-helix' structure, which is composed of 60-66 amino acids called the homeodomain (HD).The HD is encoded by specific DNA sequences that regulate target gene expression at specific times during plant development [4].In addition to the HD, all WOX members contain a WUS-box motif that regulates stem cell properties and floral meristem size.WOX proteins containing this motif may act as transcriptional repressors in plants [5].The plant WOX transcription factor family can be categorized into three groups: the WUS clade, the intermediate clade, and the ancient clade [3].The ancient clade is composed of WOX members from some green algae and land plants, while the WUS clade is made up of seed plants [6].Researchers have identified WOX families in a variety of plants in recent years.The WOX gene family in Arabidopsis consists of 15 members.AtWOX1-7 and AtWUS were clustered in the WUS clade, AtWOX8, 9, 11, and 12 were in the intermediate clade, and AtWOX10, 13, and 14 in the ancient clade [7].The genome of P. pinaster contains 14 WOX genes, while P. taeda has 11 [8]; Oryza sativa contains 13 WOX genes [9]; Zea mays contains 21 WOX genes [10]; Populus trichocarpa contains 12 real WOX genes, and Sorghum bicolor contains 11 WOX genes [11]; P. tabulaeformis contains 12 WOX [12], Picea abies contains 12 WOX [13], Ginkgo biloba contains seven WOX genes and P. sylvestris contains four WOX genes [14], and Triticum aestivum L. contains 14 WOX genes [15].In upland cotton, such as Gossypium arboreum, G. raimondii, and G. hirsutum, there are, respectively, reported counts of 21, 20, and 38 WOX genes [16].
Previous studies have reported that WOX genes are widely involved in plant responses to environmental stresses like drought, salt, cold, etc. AtHOS9 (AtWOX6) can positively regulated freezing tolerance in Arabidopsis [17].Overexpression of OsWOX11 can control root development and improve the tolerance of rice to potassium deficiency [18].The overexpression of OsWOX13, driven by the rab21 promoter, enhances drought tolerance and accelerates flowering by 7-10 days in rice.OsWOX13 directly binds to the ATTGATG motif in the promoters of drought-responsive genes OsDREB1A and OsDREB1F, thus mediating rice's response to drought stress [19].PagWOX11/12a can regulate the salt tolerance of poplar by activating the expression of PagCYP736A12 gene [20].Moreover, PagWOX11/12a can directly bind to SAUR36 and significantly promote adventitious root development and increased salt tolerance in poplar [21].The expression of PagWOX11/12a was predominantly observed in the roots of the '84K' poplar (Populus alba × P. glandulosa) and exhibited significant upregulation under drought stress conditions.Compared to the wild type, transgenic poplar that overexpressed PagWOX11/12a exhibited increased root biomass and improved drought tolerance.Conversely, poplar with inhibited PagWOX11/12a exhibited a contrasting phenotype [22].Additionally, PagWOX11/12a can regulate the level of reactive oxygen species (ROS) through modulation of ROS-related gene expression to enhance plant drought resistance [23].Overexpression of JrWOX11 in poplar enhances tolerance of NaCl and osmotic stress in transgenic poplar [24].Overexpression of JcWOX5 (Jatropha curcas) in rice has been shown to enhance plant susceptibility to drought stress [25].GhWOX4 can enhance the tolerance of drought stress by regulating the growth of the vascular system in cotton (Gossypium Hirsutum) [26].
Although the identification and function of WOX genes have been extensively studied in various species, no reports on P. massoniana are available.Therefore, we identified PmWOX genes and conducted preliminary analysis using bioinformatics methods.Subsequently, we analyzed the expression patterns of four selected WOX genes across various tissues and treatments.Moreover, we detected their transcriptional activation.This study provides insights into the role of WOX genes under abiotic stresses.

Identification and Cloning of WOX Genes in P. massoniana
We identified eight putative WOX genes from four P. massoniana transcriptomes in the absence of a reference genome and designated them in the order of identification as WOX followed by numbers in ascending order: PmWOX1-8.Moreover, we observed a high degree of conservation in the WOXX/WOX8, WUS, and WOX3/PRS1 sequences within the genomes of P. tabulaeformis, P. pinaster, and P. taeda.However, despite our efforts to blast these three genes in various transcriptomes, no significant matches were found.To investigate further, we extracted RNA from different tissues and reverse transcribed it into cDNA for use as PCR templates.Surprisingly, our PCR experiments consistently failed to clone these three genes using cDNA derived from needle, stem, or root tissues.Ultimately, we were able to clone these three genes using templates obtained from 10-day-old seedlings (Figure 1).We designated these three genes as PmWOX9, PmWUS, and PmWOXX, with lengths of 567 bp, 648 bp, and 506 bp, respectively.Through multiple sequences alignment, we found that these three genes are highly conserved within the Pinaceae family.The homology between PtPRS1 and PmWOX9, PtWOX7 and PmWOXX, and PtWUS and PmWUS is 98.26%, 98.63%, and 98.3%, respectively.We have submitted PmWOX9, PmWUS, and PmWOXX to the National Center for Biotechnology Information (NCBI), with the accession numbers PP033244, PP033245, and PP03324, respectively.
Afterward, SMART and Pfam analyses were conducted to validate the presence of conserved domains.Subsequently, we obtained a total of 11 PmWOX protein sequences, with their coding sequences provided in Table S2.Among these 11 WOX proteins, the amino acid count ranged from 80 to 482, while the predicted molecular weight varied between 9.71 and 53.15 kDa.The pI values spanned from 5.45 to 10.26.Based on predictions by CELLO, Wolf PSORT, and Plant-mPLoc, all WOX genes were predicted to be located at the nucleus.Detailed information regarding the PmWOX genes can be found in Table 1.Afterward, SMART and Pfam analyses were conducted to validate the presence of conserved domains.Subsequently, we obtained a total of 11 PmWOX protein sequences, with their coding sequences provided in Table S2.Among these 11 WOX proteins, the amino acid count ranged from 80 to 482, while the predicted molecular weight varied between 9.71 and 53.15 kDa.The pI values spanned from 5.45 to 10.26.Based on predictions by CELLO, Wolf PSORT, and Plant-mPLoc, all WOX genes were predicted to be located at the nucleus.Detailed information regarding the PmWOX genes can be found in Table 1.PmWOX7, PmWOX9, PmWOXX, and PmWUS were clustered in the WUS clade.PmWOX1-4 and PmWUS are highly conserved in gymnosperm and angiosperm plants.However, PmWOX9 and PmWOXX are highly conserved in Pinaceae.All WOX members in each group were clearly divided into gymnosperm and angiosperm subgroups.It may indicate that WOX genes are structurally and functionally differentiated during plant evolution.We chose four conserved genes, PmWOX1-4, in plants for further study.

Protein Domain Analysis of PmWOX Proteins
According to the MEME program identification of the conserved motifs of the 11 PmWOX proteins (Figure 3), we found that all proteins had highly conserved motif 1.It indicates that motif 1 is the basic structure of WOX.It is essential for WOX to perform a function.The amino acid length of the 10 motifs ranged from 6 to 50 (Table 2).All members of the ancient clade contained motif 1, motif 2, and motif 5, except PmWOX8, which lacks motif 2. We found that motif 2, motif 4, and motif 5 disappeared during WOX evolution from the ancient clade to the modern clade.Motif 1 and motif 3 are distributed in all clades.Moreover, we found that all PmWOX genes contained a typical domain 'helixloop-helix-turn-helix' (Figure 4) through multiple protein sequence alignment.

Protein Domain Analysis of PmWOX Proteins
According to the MEME program identification of the conserved motifs of the 11 PmWOX proteins (Figure 3), we found that all proteins had highly conserved motif 1.It indicates that motif 1 is the basic structure of WOX.It is essential for WOX to perform a function.The amino acid length of the 10 motifs ranged from 6 to 50 (Table 2).All members of the ancient clade contained motif 1, motif 2, and motif 5, except PmWOX8, which lacks motif 2. We found that motif 2, motif 4, and motif 5 disappeared during WOX evolution from the ancient clade to the modern clade.Motif 1 and motif 3 are distributed in all clades.Moreover, we found that all PmWOX genes contained a typical domain 'helix-loop-helix-turn-helix' (Figure 4) through multiple protein sequence alignment.

Analysis of the Transcriptional Profiles of PmWOX Genes under Drought Stress
The expression patterns under drought were validated by relative expression (qRT-PCR) and FPKM (RNA-seq) values.The heatmap of partial WOX genes was generated based on drought stress transcriptome data (Figure 5).The expression levels of some WOXs in the transcription group under drought stress were too low to be detected, so only six genes were given.The heatmap showed the expressions of partial genes under different field capacity levels.The expression of PmWOX1 tended to decrease as the degree of drought deepened and increased in severe drought; all of them showed lower expression compared to CK.The expression of PmWOX2 increased slightly in mild drought and decreased under medium and severe drought.The expression of PmWOX3, PmWOX5, and PmWOX6 continued to increase as the drought deepened.On the contrary, the expression of PmWOX4 was inhibited by drought.All of them showed lower expression compared to CK.Moreover, we selected four target genes for further verification by qRT-PCR.When P. massoniana was subjected to water loss conditions, the expression of PmWOX2 decreased gradually and reached its lowest level at 7 days, followed by no significant change at 12 and 20 days.Conversely, the expression of PmWOX1 and PmWOX3 exhibited a significant increase at 12 days.Additionally, the expression of PmWOX4 initially decreased within the first 12 days but peaked at 20 days.Due to the disparities in sample sources and sampling time between qRT-PCR and transcriptome sequencing, as well as the individual seedling used for qRT-PCR analysis compared to the pooled samples used in transcriptome sequencing, certain inconsistencies may arise.However, the expression trends of the four selected genes at T1, T2, and T3 are consistent with the trends at 3, 7, and 12 d.
WOXs in the transcription group under drought stress were too low to be detected, so only six genes were given.The heatmap showed the expressions of partial genes under different field capacity levels.The expression of PmWOX1 tended to decrease as the degree of drought deepened and increased in severe drought; all of them showed lower expression compared to CK.The expression of PmWOX2 increased slightly in mild drought and decreased under medium and severe drought.The expression of PmWOX3, PmWOX5, and PmWOX6 continued to increase as the drought deepened.On the contrary, the expression of PmWOX4 was inhibited by drought.All of them showed lower expression compared to CK.Moreover, we selected four target genes for further verification by qRT-PCR.When P. massoniana was subjected to water loss conditions, the expression of PmWOX2 decreased gradually and reached its lowest level at 7 days, followed by no significant change at 12 and 20 days.Conversely, the expression of PmWOX1 and PmWOX3 exhibited a significant increase at 12 days.Additionally, the expression of PmWOX4 initially decreased within the first 12 days but peaked at 20 days.Due to the disparities in sample sources and sampling time between qRT-PCR and transcriptome sequencing, as well as the individual seedling used for qRT-PCR analysis compared to the pooled samples used in transcriptome sequencing, certain inconsistencies may arise.However, the expression trends of the four selected genes at T1, T2, and T3 are consistent with the trends at 3, 7, and 12 d.

Expression Patterns of PmWOX Genes in Different Tissues
The qRT-PCR results in Figure 6 showed the expression patterns of four PmWOX genes across eleven distinct tissues: shoot apices (T); young needle (YN); old needle (ON); young stem (YS); old stem (OS); xylem (X); phloem (P); root (R); callus (C); seed (S); young seedling (YSe).It is evident from the results that PmWOX1 exhibits ubiquitous expression across all tissues, with particularly high levels observed in young needles and relatively low levels detected in roots, calluses, seeds, and young seedlings.Notably, PmWOX2 displays significantly higher expression in callus compared to other tissues; moreover, its expression is almost undetectable in seeds and young seedlings.Among all tissues examined, PmWOX3 demonstrates the highest expression levels in the callus followed by phloem and root; conversely, its expression is considerably lower in seeds and young seedlings.Similarly expressed across all tissues examined, PmWOX4 exhibits notably high expression levels in needles, callus, and xylem, while displaying relatively low levels specifically within young seedlings; no significant differences were observed among other tissues.Overall, three genes exhibit elevated expressions within the callus except for PmWOX1, and all of them did not tend to be expressed in seeds and young seedlings.
genes across eleven distinct tissues: shoot apices (T); young needle (YN); old needle (ON); young stem (YS); old stem (OS); xylem (X); phloem (P); root (R); callus (C); seed (S); young seedling (YSe).It is evident from the results that PmWOX1 exhibits ubiquitous expression across all tissues, with particularly high levels observed in young needles and relatively low levels detected in roots, calluses, seeds, and young seedlings.Notably, PmWOX2 displays significantly higher expression in callus compared to other tissues; moreover, its expression is almost undetectable in seeds and young seedlings.Among all tissues examined, PmWOX3 demonstrates the highest expression levels in the callus followed by phloem and root; conversely, its expression is considerably lower in seeds and young seedlings.Similarly expressed across all tissues examined, PmWOX4 exhibits notably high expression levels in needles, callus, and xylem, while displaying relatively low levels specifically within young seedlings; no significant differences were observed among other tissues.Overall, three genes exhibit elevated expressions within the callus except for PmWOX1, and all of them did not tend to be expressed in seeds and young seedlings.The highest column is marked with 'a', then 'b' and so on.Completely different lowercase letters between different columns indicate a significant difference, p < 0.05.More than one lowercase letter in the same column indicate no significant difference between the column and other columns that contain one of the lowercase letters.The relative expression in "T"was set as 1.

Expression Patterns of PmWOX Genes under Different Treatments
The qRT-PCR results presented in Figure 7 demonstrate the expression patterns of four PmWOX genes under various treatments.Specifically, the expressions of PmWOX1 and PmWOX3 were induced significantly by ABA at 6 h; while PmWOX2 and PmWOX4 exhibited lower expression levels under ABA treatment.Furthermore, PmWOX1, PmWOX2, and PmWOX4 can be induced at different time points by MeJA.However,

Expression Patterns of PmWOX Genes under Different Treatments
The qRT-PCR results presented in Figure 7 demonstrate the expression patterns of four PmWOX genes under various treatments.Specifically, the expressions of PmWOX1 and PmWOX3 were induced significantly by ABA at 6 h; while PmWOX2 and PmWOX4 exhibited lower expression levels under ABA treatment.Furthermore, PmWOX1, PmWOX2, and PmWOX4 can be induced at different time points by MeJA.However, MeJA inhibited the expression of PmWOX3.Notably, the expression of PmWOX1 increased significantly at 6 h but decreased gradually thereafter under SA treatment.The expression of PmWOX2 decreased significantly at 12 h, whereas that of PmWOX3 peaked at 24 h.Under SA treatment, the expression level of only one gene (PmWOX4) continued to decrease over time.Additionally, ETH significantly inhibited both PmWOX2 and PmWOX3 expressions; however, other genes did not show sensitivity to ETH treatment.Moreover, H 2 O 2 treatment led to a significant increase in the expression level of PmWOX1 after 12 h, but caused a notable decrease in PmWOX2 and PmWOX3 expressions after this time point.Furthermore, the presence of PEG resulted in the inhibition of all four genes after 6 h.PmWOX1, 2, and 4 were induced by NaCl, while PmWOX3 did not show a significant change.In addition, PmWOX1 and PmWOX3 showed a continuous decrease in expression following injury to P. massoniana.The expression of PmWOX2 and PmWOX4 increased at 3 h but subsequently decreased.difference between the column and other columns that contain one of the lowercase letters.The relative expression in "0" was set as 1.

Transcriptional Activation Activity
The yeast growth assay photographs (Figure 8) revealed that only yeast with pGBKT7-PmWOX3 was able to grow on SD/-Trp/-His/-Ade medium and exhibited a blue Completely different lowercase letters between different columns indicate a significant difference, p < 0.05.More than one lowercase letter in the same column indicate no significant difference between the column and other columns that contain one of the lowercase letters.The relative expression in "0" was set as 1.

Transcriptional Activation Activity
The yeast growth assay photographs (Figure 8) revealed that only yeast with pGBKT7-PmWOX3 was able to grow on SD/-Trp/-His/-Ade medium and exhibited a blue color, while the other three failed to support yeast growth on SD/-Trp/-His/-Ade medium.These results unequivocally demonstrated that only PmWOX3 exhibited transcriptional activation, whereas the remaining three genes did not exhibit any self-activation.The results suggested that PmWOX3 can function as a transcriptional regulator in P. massoniana.
SA, (d) ETH, (e) H2O2, (f) PEG, (g) NaCl, (h)Injury.The same lowercase letters between d columns indicate no significant difference.The highest column is marked with 'a', then 'b' on.Completely different lowercase letters between different columns indicate a sign difference, p < 0.05.More than one lowercase letter in the same column indicate no sign difference between the column and other columns that contain one of the lowercase lette relative expression in "0" was set as 1.

Transcriptional Activation Activity
The yeast growth assay photographs (Figure 8) revealed that only yeas pGBKT7-PmWOX3 was able to grow on SD/-Trp/-His/-Ade medium and exhibited color, while the other three failed to support yeast growth on SD/-Trp/-His/-Ade me These results unequivocally demonstrated that only PmWOX3 exhibited transcrip activation, whereas the remaining three genes did not exhibit any self-activatio results suggested that PmWOX3 can function as a transcriptional regulator massoniana.

Discussion
The WOX family, known as plant-specific TFs, plays an important role in regu plant growth, development, and abiotic stress.In recent years, the identification of transcription factor families has been reported in various plants.However, most studies on the functions of the WOX genes have been focused on model plants lik and Arabidopsis [3].The identities and expression patterns of WOX genes invol response to abiotic stresses in P. massoniana remain unknown.Therefore, we ide WOX genes in P. massoniana, and selected four genes for further functional analysis different abiotic stresses.
In our study, we identified 11 WOX genes in P. massoniana and divided them three clades, termed the WUS, intermediate, and ancient clade (Figure 2).Further the WUS clade had the largest number of members, similar to rice, Arabidopsis [3], [11], and cotto [16].The WUS clade exhibited the largest number of memb P.massoniana and other plant species, thereby confirming its high conservation as re in previous studies.Moreover, we found that PmWUS, PmWOX9, and PmWOXX isolated from young seedlings rather than mature tissues.We hypothesized that, embryo matures into a seedling, their expression levels gradually decline and even become undetectable; they may perform specific functions in early developmen

Discussion
The WOX family, known as plant-specific TFs, plays an important role in regulating plant growth, development, and abiotic stress.In recent years, the identification of WOX transcription factor families has been reported in various plants.However, most of the studies on the functions of the WOX genes have been focused on model plants like rice and Arabidopsis [3].The identities and expression patterns of WOX genes involved in response to abiotic stresses in P. massoniana remain unknown.Therefore, we identified WOX genes in P. massoniana, and selected four genes for further functional analysis under different abiotic stresses.
In our study, we identified 11 WOX genes in P. massoniana and divided them into three clades, termed the WUS, intermediate, and ancient clade (Figure 2).Furthermore, the WUS clade had the largest number of members, similar to rice, Arabidopsis [3], poplar [11], and cotto [16].The WUS clade exhibited the largest number of members in P.massoniana and other plant species, thereby confirming its high conservation as reported in previous studies.Moreover, we found that PmWUS, PmWOX9, and PmWOXX can be isolated from young seedlings rather than mature tissues.We hypothesized that, as the embryo matures into a seedling, their expression levels gradually decline and eventually become undetectable; they may perform specific functions in early development of P. massoniana.This is consistent with what has been reported in other plants.For example, AtWOX5 is expressed in the quiescent center of the root tip, but shows low or undetectable expression levels in other regions [27].During embryogenesis, PaWOX2 exhibits the highest expression at the early development stages, but low levels were detected in seedling tissues [28].PpWOX3, PpWUS, and PpWOXX were expressed in mature embryos.Specifically, PpWUS was expressed highly in 1-2 cm germinated embryos and lowly in 3 cm embryos.Compared to mature embryos, PpWOX3 and PpWOXX show lower expression levels in germinated embryos [8].The expression of PpWUS, PpWOXX, and PpWOX3 is predominantly observed in the shoot apex of 3-week-old seedlings, while remaining undetectable in other tissues of the seedling.
The gene expression profile is correlated with their respective functions.We detected the expression levels of PmWOX1-4 in different tissues.The results indicated their involvement in the growth and development processes at various stages in P. massoniana.The high expression observed in callus suggested that PmWOX2 and PmWOX3 may play an important role during the process of callus development, while also maintaining their involvement in the growth and development of P. massoniana throughout maturation.The transformation of gymnosperms poses significant challenges, primarily due to the difficulty in establishing a regeneration system.This is attributed to the challenging nature of callus induction, maintenance, and proliferation.The functions of PmWOX2, PmWOX3, PmWOX9, PmWOXX, and PmWUS, which were expressed in callus or at specific stages of embryo development, remain to be analyzed in the future to resolve this issue.
Proteins must be localized at the appropriate region to perform their function.Subcellular localization prediction results indicated that WOX TFs likely exert regulatory functions at the nucleus, which was consistent with WOX gene localization in Arabidopsis [29][30][31][32], rice [33,34], Populus tomentosa [35], and Rosaceae [36].Regulatory responses to abiotic stress require several important metabolic proteins, such as osmoprotective proteins and regulatory proteins, as well as protein kinases and TFs involved in transduction in signal transduction pathways [37].Some WOX genes can bind to stress-related genes to resist external stresses, such as OsWOX13 [19] and PagWOX11/12a [20].In addition, PmWOX3 exhibited transcriptional activation and further exploration of its downstream regulated genes can provide insights into its molecular mechanisms involved in stress resistance and development of P. massoniana.For example, OsWOX3 in rice can inhibit the expression of YAB3 and regulate the development of rice leaves [38].During early embryonic development, AtWOX8, together with its functional overlap gene AtWOX9, is activated by WRKY2 transcription factor in the zygote, its basal daughter cell, and the hypophysis [39].
An increasing body of research demonstrates the involvement of WOX genes in the response to diverse abiotic stresses across various plant species [16,37].WOX genes can show different responses when plants are under abiotic stresses.For, example, drought or salt stress can either up-regulate or down-regulate the transcriptional abundance of some WOX genes in soybean [37].Similarly, we found that PmWOX1 was significantly induced by ABA, MeJA, H 2 O 2 , and drought stress, indicating that PmWOX1 may play an important role in responding to abiotic stresses.On the other hand, PmWOX2 could respond to salt stress and MeJA treatment.PmWOX3 showed significant induction during drought stress and did not exhibit sensitivity toward salt stress.Furthermore, while PmWOX4 was non-sensitive to ETH, it could be induced by NaCl, injury, drought, and MeJA.PmWOX1, PmWOX3, and PmWOX4 showed significant response to drought, as reported in rice [18], poplar [22], and cotton [26].
Numerous abiotic stresses lead to the generation of reactive oxygen species (ROS) in plants [40].Excessive accumulation of ROS engenders oxidative stress, culminating in cell death and even plant death [41].Consequently, plants undergo physiological and biochemical alterations that facilitate their manifestation of stress tolerance [42].In summary, our findings suggest that WOX genes may play crucial roles in regulating P. massoniana under abiotic stress conditions.This study could provide valuable insights for subsequent functional gene studies.However, further genetic verification is warranted to elucidate their precise mechanisms.

Plant Materials and Abiotic Stress Treatments
Two-year-old P. massoniana seedlings were obtained from the State Key Laboratory of Tree Genetics and Breeding (Nanjing Forestry University).Callus was induced by a mature embryo of P. massoniana.Eight representative tissues (shoot apices; young needle; old needle; young stem; old stem; xylem; phloem and root) were collected from 3 two-year-old seedlings of the same height in the same pot.Whole young seedlings were collected 10 days after sowing.The two-year-old seedlings were treated with 9 abiotic stresses.The drought treatment was performed following the following method: collect samples five times in 20 days through natural evaporation after watering at 0 days, the field capacity being as follows: 0 d (67%); 3 d (63%); 7 d (58%); 12 d (46%); 20 d (34%).The mechanical damage treatment method was performed by cutting the upper half of the needles.The osmotic stress was induced by soaking the plants in the 15% polyethylene glycol (PEG6000) solution and 200 mM NaCl solution.For plant hormone treatment, the selected seedlings were sprayed independently with 100 µM ABA (abscisic acid); 1 mM SA (salicylic acid); 10 mM H 2 O 2 (hydrogen peroxide); 10 mM MeJA (methyl jasmonate); 50 µM ETH (ethephon) solutions (50 mL) on the surface of needles.Afterward, needles were sampled at 0 h, 3 h, 6 h, 12 h, and 24 h after treatment, except in the case of drought stress.The needles under drought stress were sampled at 0 d, 3 d, 7 d, 12 d, and 20 d.All treatments were conducted on three biological replicates and three technological replicates.

RNA Extraction and qRT-PCR Analysis
Total RNA was extracted using the FastPure Plant Total RNA Isolation Kit (RC401, Vazyme Biotech, Nanjing, China).RNA concentration and purity were measured with a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA), and RNA integrity was estimated by 1% agarose gel electrophoresis.First-strand cDNA was synthesized using the One-step gDNA Removal and cDNA Synthesis Kit (AT311, TransGen Biotech, Beijing, China).Primers for quantitative real-time reverse transcription PCR (qRT-PCR) were designed using Primer 5.0 (Table S1).SYBR Green reagents were used to detect the target sequence.Each PCR mixture (10 µL) contained 1 µL of diluted cDNA (20× dilution), 5 µL of SYBR Green Master Mix (11184ES03, Yeasen Biotech, Shanghai, China), 0.4 µL of each primer (10 µM), and 3.2 µL of ddH 2 O.The PCR program stages were: (1) 95 • C for 2 min (preincubation); (2) 95 • C for 10 s, (3) 60 • C for 30 s, repeated 40 times; the remaining steps used the instrument default settings.The PCR quality was estimated based on melting curves.Alpha-tubulin (TUA) gene was used as a reference gene [45].Three independent biological replicates and three technical replicates for each biological replicate were examined.Quantification was achieved using comparative cycle threshold (Ct) values, and gene expression levels were calculated as 2 ( −∆∆Ct ) [∆CT = CT Target − CT TUA.∆∆Ct = ∆Ct Target − ∆Ct CK].Duncan's test was used to examine the significance between different columns in IBM SPSS Statistics (Version 25).The results were marked with lowercase letters, starting with the largest average.The same lowercase letters between different columns indicate no significant difference.Completely different lowercase letters between different columns indicate a significant difference, c < 0.05.More than one lowercase letter in the same column indicate no significant difference between the column and other columns that contain one of the lowercase letters.

Transcriptional-Activation Activity Assay
We constructed recombinant vectors pGBKT7-PmWOX containing the complete open reading frame (ORF) sequences of PmWOX1, PmWOX2, PmWOX3, and PmWOX4 to investigate their potential for transcriptional self-activation.The primer sequences used for vector construction are provided in Table S1.The empty pGBKT7 vector was used as a negative control, and PmC3H20 which was validated previously [46] was used as a positive control.Subsequently, these fusion vectors were transformed into yeast strain AH109 (YC1010, Weidi Biotechnology, Shanghai, China) according to the operational approach.Transformed yeast strains were screened on selective YPDA medium plates lacking tryptophan (SD/-Trp), and cultivated at 29 • C for 48 h.Single yeast colonies were collected in 10 µL ddH 2 O and confirmed by PCR analysis.Upon positive detection, the remaining positive bacterium liquid was diluted to a volume of 200 uL with ddH 2 O; subsequently, 5 µL of this dilution was plated onto different yeast media, lacking tryptophan/histidine (SD/-Trp/-His) and tryptophan/histidine/leucine (SD/-Trp/-His/-Ade), respectively.The later medium was supplemented with X-α-Gal on the surface to enable color development.Finally, photographic documentation was performed to record yeast growth.

Conclusions
In this study, we initially identified eight PmWOX genes in P. massoniana, and subsequently cloned three WOX genes based on sequences from other Pinaceae plants.Bioinformatics analysis was performed to further investigate their characteristics.Phylogenetic analysis revealed that all WOX members were categorized into three distinct clades, while the presence of a 'helix-loop-helix-turn-helix' structure was observed in all WOX genes.Notably, PmWOX1-4 exhibit distinct responses to various stress conditions, whereas the expression of PmWOX2-4 in callus suggested their potential roles in callus development.Furthermore, due to its transcriptional activation ability, PmWOX3 could be considered a key gene for subsequent functional verification studies.Additionally, considering its significant expression in the callus, PmWOX2 is an ideal candidate for investigating callus development specifically within P. massoniana.PmWOX1 can be induced by ABA, ROS, and drought, suggesting its potential as a candidate gene for further investigation into its abiotic stress resistance.Importantly, this study represents the first report on WOX genes in P.massoniana, and provides valuable insights into the expression patterns of PmWOX genes.This study lays the foundation for future research on WOX genes, not only within P. massoniana but also across other related taxa.

Figure 4 .
Figure 4. Alignment of the conserved region with PmWOX proteins.

Figure 4 .
Figure 4. Alignment of the conserved region with PmWOX proteins.

Figure 4 .
Figure 4. Alignment of the conserved region with PmWOX proteins.

Figure 5 .
Figure 5. (a) Transcriptional profiles of WOX genes in P. massoniana under different drought levels: CK (80 ± 5)%, T1 (65 ± 5)%, T2 (50 ± 5)%, and T3 (35 ± 5)%.A heatmap was generated using log2 (FPKM + 1) values, then normalized by row scale.The color scale represents relative expression levels.(b) Relative expression of PmWOX1-4 genes under drought stress.The same lowercase letters between different columns indicate no significant difference.The highest column is marked with 'a', then 'b' and so on.Completely different lowercase letters between different columns indicate a significant difference, p < 0.05.More than one lowercase letter in the same column indicates no significant difference between the column and other columns that contain one of the lowercase letters."The relative expression in "0" was set as 1.

Figure 5 .
Figure 5. (a) Transcriptional profiles of WOX genes in P. massoniana under different drought levels: CK (80 ± 5)%, T1 (65 ± 5)%, T2 (50 ± 5)%, and T3 (35 ± 5)%.A heatmap was generated using log2 (FPKM + 1) values, then normalized by row scale.The color scale represents relative expression levels.(b) Relative expression of PmWOX1-4 genes under drought stress.The same lowercase letters between different columns indicate no significant difference.The highest column is marked with 'a', then 'b' and so on.Completely different lowercase letters between different columns indicate a significant difference, p < 0.05.More than one lowercase letter in the same column indicates no significant difference between the column and other columns that contain one of the lowercase letters."The relative expression in "0" was set as 1.

Figure 6 .
Figure 6.Relative expression of PmWOX1-4 genes in different tissues.T: Shoot apices; YN: young needle; ON: old needle; YS: young stem; OS: old stem; X: xylem; P: phloem; R: root; C: callus; S: seed; YSe: young seedling.The same lowercase letters between different columns indicate no significant difference.The highest column is marked with 'a', then 'b' and so on.Completely different lowercase letters between different columns indicate a significant difference, p < 0.05.More than one lowercase letter in the same column indicate no significant difference between the column and other columns that contain one of the lowercase letters.The relative expression in "T"was set as 1.

Figure 6 .
Figure 6.Relative expression of PmWOX1-4 genes in different tissues.T: Shoot apices; YN: young needle; ON: old needle; YS: young stem; OS: old stem; X: xylem; P: phloem; R: root; C: callus; S: seed; YSe: young seedling.The same lowercase letters between different columns indicate no significant difference.The highest column is marked with 'a', then 'b' and so on.Completely different lowercase letters between different columns indicate a significant difference, p < 0.05.More than one lowercase letter in the same column indicate no significant difference between the column and other columns that contain one of the lowercase letters.The relative expression in "T"was set as 1.

Figure 7 .
Figure 7. Relative expressions of PmWOX genes under different treatments.(a) ABA, (b) MeJA, (c) SA, (d) ETH, (e) H2O2, (f) PEG, (g) NaCl, (h)Injury.The same lowercase letters between different columns indicate no significant difference.The highest column is marked with 'a', then 'b' and so on.Completely different lowercase letters between different columns indicate a significant difference, p < 0.05.More than one lowercase letter in the same column indicate no significant

Figure 7 .
Figure 7. Relative expressions of PmWOX genes under different treatments.(a) ABA, (b) MeJA, (c) SA, (d) ETH, (e) H 2 O 2 , (f) PEG, (g) NaCl, (h) Injury.The same lowercase letters between different columns indicate no significant difference.The highest column is marked with 'a', then 'b' and so on.Completely different lowercase letters between different columns indicate a significant difference, p < 0.05.More than one lowercase letter in the same column indicate no significant difference between the column and other columns that contain one of the lowercase letters.The relative expression in "0" was set as 1.

Figure 8 .
Figure 8. Transcriptional activation assay of four PmWOX genes.An empty pGBKT7 vect used as a negative control.PmC3H20 was used as a positive control.

Figure 8 .
Figure 8. Transcriptional activation assay of four PmWOX genes.An empty pGBKT7 vector was used as a negative control.PmC3H20 was used as a positive control.

Table 1 .
Detailed information and physicochemical properties of PmWOXs.

Table 1 .
Detailed information and physicochemical properties of PmWOXs.

Protein ID Prediction of Subcellular Localization Amino Acid Number (aa) Protein Molecular Weight (KD) Isoelectric Point Instability Index Grand Average of Hydropathicity
PmWOXX, and PmWUS were clustered in the WUS clade.PmWOX1-4 and PmWUS are highly conserved in gymnosperm and angiosperm plants.However, PmWOX9 and PmWOXX are highly conserved in Pinaceae.All WOX members in each group were clearly divided into gymnosperm and angiosperm subgroups.It may indicate that WOX genes are structurally and functionally differentiated during plant evolution.We chose four conserved genes, PmWOX1-4, in plants for further study.

Table 2 .
The sequences of conserved motifs.

Table 2 .
The sequences of conserved motifs.

Table 2 .
The sequences of conserved motifs.